Investigation into the effect of optogenetic stimulation on the structure and function of stem cell derived cardiomyocytes

[摘要] Introduction: The first beats of the developing heart resemble peristaltic contractions with electrical activity arising in the precursor to the sino-atrial node and propagating through the heart tube. The purpose of this thesis was to investigate channelrhodopsin-2 (ChR-2) based optogenetic stimulation of induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with the intention of using the technique to investigate the functional effects of directional activation on immature cardiomyocytes.Methods: Commercially supplied iPSC-CMs (Axiogenesis) were made to express ChR-2 (H134R) via an adeno-associated viral vector (AAV1.CAG.hChR2(H134R)-mCherry.WPRE.SV40). Cell function was assessed before and after pacing using in house software to measure contraction kinetics and strength-duration curves to determine cell excitability. α-actinin staining allowed for analysis of sarcomere length, inhomogeneity and alignment.Results: Commercially supplied iPSC-CMs had significantly shorter sarcomeres than their adult counterparts (1.766 ± 0.017 μm vs 2.028 ± 0.079 μm respectively, P=0.0125 unpaired t-test). A viral load of 2500GC/cell produced ChR-2 expression sufficient for optical control of these cells 5 days post transfection. The chronaxie of photostimulated cells (1.53 ± 0.32 ms) did not differ significantly from that of electrically stimulated cells (2.17 ± 0.44 ms, P=0.2434 unpaired t-test). Prolonged photostimulation of a single site led to a loss of stimulation:contraction coupling after 2.22 ± 1.26 hours and almost complete loss of contractile function after 24 hours. Alternating stimulation between multiple sites allowed cells to be paced for 96 hours and allowed directional electrical activation to be imposed on immature heart cells. 96 hours of directional pacing produced no changes in cell structure or cell excitability but produced a significant reduction in contraction upstroke time (72.03 ms vs 48.85 ms P=0.023 one-way ANOVA) 24 hours after the termination of stimulation.Conclusions: This project has demonstrated that replicating the directional electrical activity of the developing heart can induce a modest change in contractile function that may represent a step towards functional maturation of commercially available iPSC-CMs.

[发布机构] University:University of Glasgow;Department:Institute of Cardiovascular and Medical Sciences

[关键词] Stem cell derived cardiomyocytes, iPSC-CMs, optogenetics, channelrhodopsin, chronic pacing.